Recent work by the applicant and others has identified an electrogenic proton pump (H+-ATPase) in rat liver endocytic vesicles and in organelles in other tissues. This H+-ATPase plays an important role in receptor-mediated endocytosis and urinary acidification, yet little is known regarding its regulation or role in other cell functions. The proposed studies will further characterize the H+-ATPase in rat liver endocytic vesicles and determine its role in other liver processes. The specific aims of this proposal are (1) to study in detail the function of H+-ATPase and its possible regulation by anions and organic nitrates; (2) to characterize transport of C1- (essential for vesicle acidification) in the same vesicles; (3) to quantitate pH and membrane potential gradients established by H+-ATPase and to examine these aspects of H+-ATPase function in vesicles derived from four different steps along the endocytic pathway; (4) to explore a possible plasma membrane location for H+-ATPase and to determine its role in hepatocyte pHi regulation, H+ efflux, biliary HCO3- secretion, and bile acid-independent bile formation (BAIBF); and (5) to explore the hypothesis that hepatocyte H+/HCO3- transport and BAIBF may be regulated, in part, by exocytotic insertion/endocytic removal of H+-ATPase units into/from the plasma membrane. These specific aims will be addressed using a variety of experimental models and techniques. Fluorescent probes and radiolabeled tracers will be used to examine and quantitate H+-ATPase function in intracellular and plasma membrane vesicles. pHi regulation and H+ efflux will be studied in isolated hepatocytes using fluorescent and pH-STAT techniques. Bile formation and endocytosis will be studied in the isolated perfused rat liver and in cultured rat hepatocytes. All of these methods are currently in use in the applicant's laboratory, and the ability to use a variety of models and techniques and to integrate the findings from studies at the subcellular, cellular, and intact organ level will facilitate attainment of these specific aims. The H+-ATPase appears to be widely distributed in nature, yet these issues have not been systematically addressed in any cell type. Thus, the findings of the proposed studies are expected to shed new light not only on liver endocytosis, pHi regulation, bile formation, and cholestasis, but also on pHi regulation, endocytosis, lysosomal degradation, cholesterol metabolism, storage and secretion of neurotransmitters, hormones and proteins, viral infection and epithelial H+ transport in many cell types.